Immunotoxins (ITs) are recombinant fusion proteins originally developed for the treatment of malignant diseases. They comprise a toxic effector domain fused to a tumor cell-specific binding component, which is usually an antibody or a derivative thereof. Initially the antibody components were derived from mice and the toxins were derived from bacteria or plants, e.g. Pseudomonas aeruginosa exotoxin A (ETA), diphtheria toxin (DT) or ricin A. The major advantage of ITs compared to traditional chemotherapy is their exceptional target cell specificity, but their disadvantages include side effects caused by potential immunogenicity. Furthermore, the development of neutralizing antibodies against the toxins or murine parts of binding moieties limits the impact of long-term treatment due to accelerated clearance from the circulation. Highly toxic drugs and heterologous toxins conjugated to antibodies have been extensively tested, but for example the ADC gemtuzumab ozogamicin (Mylotarg®), approved by the US Food and Drug Administration for treatment of CD33+ acute myeloid leukemia was voluntarily withdrawn from the market because of increased occurrence of fatalities caused by hepato-occlusive disease upon treatment.
In order to circumvent the side effects caused by potential immunogenicity, the murine antibody components have been replaced by humanized or fully human counterparts and the bacterial and plant toxins have been replaced by human pro-apoptotic enzymes such as granzymes or ribonucleases (RNases). Granzyme B activates the caspase cascade whereas the RNase angiogenin (Ang) cleaves tRNA, both events leading to the induction of apoptosis. Fusion proteins that consist of a disease-specific binding component (e.g. scFvs, cytokines or peptide ligands) fused to a human pro-apoptotic enzyme are known as ‘human cytolytic fusion proteins’ (hCFPs) and have already proven their potential in several applications (Stahnke, B., et al., Mol Cancer Ther, 2008. 7(9): p. 2924-32; Huhn, M., et al., Cancer Res, 2001. 61(24): p. 8737-42; Krauss, J., et al., Br J Haematol, 2005. 128(5): p. 602-9; Schiffer, S., et al., Antibodies, 2013. 2(1): p. 9-18).
Nevertheless, the improvement of hCFPs is necessary because their cytotoxic efficacy is much lower than that of conventional ITs. Consequently, higher doses are needed to achieve the same therapeutic effect and the risk of inducing a treatment-associated immune response is therefore greater. The lower efficacy of hCFPs may also reflect the absence of natural translocation domains in human pro-apoptotic proteins. These are present in bacterial and plant toxins and promote endosomal release, so that the cytotoxic enzymes are delivered into the cytosol. Artificial adapter peptides have recently been designed to promote the release of hCFPs from endosomes and by this increase their cytotoxic efficacy.
The generation of RNase angiogenin (Ang)-based hCFPs and the cell-specific elimination of pathogenic cell populations worked successful in several studies so far. Nevertheless, the IC50 of angiogenin-based hCFPs is significantly higher on different cell lines than for ITs containing ETA, diphtheria toxin or ricin, which are generally in the picomolar range (Ribbert, T., et al., Br J Dermatol, 2010. 163(2): p. 279-86; Zhong, R. K., et al., J Hematother Stem Cell Res, 2001. 10(1): p. 95-105). The greatest limitation affecting the inhibition of protein biosynthesis by angiogenin is the presence of its natural inhibitor RNH1 (also known as RI, RNH or PRI). This protein has a horseshoe-shaped structure that binds all monomeric members of the pancreatic RNase family with extraordinary affinity (De Lorenzo, C., et al., FEBS Lett, 2007. 581(2): p. 296-300). It is one of the strongest protein interactions discovered thus far, with a Ki≈10−13-10−16 for angiogenin, which helps to protect cellular nucleic acids against invading secretory RNases. The RNase-inhibitor complex has a half-life of more than 24 h, which corresponds with the low Ki value and completely inhibits the ribonucleolytic and angiogenic activity of Ang. RNH1 is continuously expressed in all human cell types and its abundance is ≥0.01% of the total intracellular protein content. It is predominantly located in the cytosol but is also found in mitochondria and the nucleus where it is thought to participate in the oxidative stress response. The cytotoxic activity of hCFPs based on RNases is therefore dependent on the delivery of sufficient amounts of RNase to overcome inhibition by RNH1 in the cytosol.
An additional limitation of angiogenin as a human CFP effector domain is its weak enzymatic activity towards standard RNase substrates compared to other members of the pancreatic RNase superfamily (Russo, N., et al., Proc Natl Acad Sci USA, 1994. 91(8): p. 2920-4).
Furthermore, human pancreatic RNase nor RI evasive variants thereof proved to be suitable as effector components for a therapeutic IgG antibody platform. Further, there are also doubts about whether the effector mechanism of “cytotoxic” RNases is only dependent on their ribonucleolytic activity because there is no correlation between catalytic efficiency of different RNases and their cytotoxic properties, suggesting more complex mechanisms than unspecific RNA cleavage (Schirrmann, T., et al., MAbs, 2014. 6(2): p. 367-80). Rather, the cytosolic delivery of ribonucleases, which is among dependent on the used scFv and the intracellular routing taking place after CFP-antigen complex internalization, seems to be crucial. Thus the chosen scFv, in combination with the ribonuclease, has to accomplish several requirements to acquire CFP cytotoxicity that also includes the shunning of endosome fusion with intracellular lysosomal compartments if no translocation domains are explicitly available.
Therefore, the availability of novel and improved human cytolytic fusion proteins (hCFPs) would be highly advantageous.